Interleukin 1 (IL-1) is a general name for two distinct proteins, IL-1 alpha and IL-1 beta, which are major pro-inflammatory cytokines. IL-1 exerts its effects by binding to specific transmembrane receptors (IL-1RI) on multiple cell types. The effects of IL-1 are counteracted by natural inhibitors such as soluble IL-1 receptors and IL-1R antagonist protein (IL1RA or IL1Ra). IL1RA inhibits the effect of IL-1 by blocking its interaction with the cell surface receptors.
Therapeutic approaches for targeting IL-1 for anti-inflammatory purposes have been addressed in the art. These include administration of recombinant IL-1R antagonist protein, IL-1 trap fusion proteins, anti-IL-1 antibodies, anti-IL-1RI and soluble IL-1RI and II in experimental models of arthritis (reviewed in Gabay C et al. 2010).
Peptides with IL-1R antagonist activity are disclosed in e.g. US20060094663A1. These sequences are derived from IL-1RAcP (IL-1RI accessory protein).
Recombinant IL-1R antagonist protein for use as an anti-inflammatory drug has been commercialised: Anakinra, sold under the trade name ‘Kinerer’ (See U.S. Pat. No. 5,075,222). It has been approved for treatment of rheumatoid arthritis. The drawbacks of anakinra is that (1) it is delivered as an injection concentrate with 100 mg in each dose; (2) it is prepared from genetically modified E. coli using recombinant DNA technology; and (3) it has a high molecular weight (corresponding to full-length IL-1RA)
Thus, identification of shorter and potent peptides derived from IL1RA may address these disadvantages, in that (1) a lower concentration of the present peptides may be used, (2) the smaller peptides are stable in solution and can be more easily chemically synthesised with a lower associated cost, and (3) the lower molecular weight of the small mimetic peptides enable them to more easily pass the blood-brain barrier, meaning a lower peptide amount is needed to reach working concentrations in the brain—this makes them particularly useful also for treatment of neuroinflammatory diseases. Specific targeting also has the potential of fewer side-effects and improved efficacy.
The WO05086695A2 patent family discloses specific peptide fragments of the IL-1R antagonist protein. These fragments are capable of inhibiting tissue destruction in inflammatory disorders, and may be used to treat chronic inflammatory disorders and rheumatoid arthritis (US2007027082; patent application of issued U.S. Pat. No. 7,674,464). No effect on neurodegenerative disorders is addressed.
According to US2007027082, the disclosed peptide fragments preferably comprise the subsequence LVAGY (“SEQ ID NO:42”); being present in “SEQ ID NOs:13, 18, 21, 23, 24 and 43” of US2007027082. Reversal of IL-1 induced effects were observed for “SEQ ID NO:13, 15, 23 and 24” in vitro (Example 3), and “SEQ ID NO:18 and 43” in vivo (Example 10).
“SEQ ID NOs:13, 18 and 19” of US2007027082 further comprise the subsequence SGRKSSKMQA of ILR1A (present SEQ ID NO:1). The shortest peptide comprising the subsequence SGRKSSKMQA of ILR1A having an effect according to US2007027082 is 35 amino acids long (“SEQ ID NO:13”), being 42 amino acids long when a nuclear localisation signal is added for optimisation (“SEQ ID NO:18”). When examined as short as 15 amino acids—excluding the LVAGY subsequence (“SEQ ID NO:19”), no effect is observed on inhibiting the collagenase production stimulated by IL-1 in vitro (Example 3). It is thus concluded in US2007027082 that all peptides active in inhibiting MMP-1 (a collagenase) by IL-1 beta contain residues LVAGY of IL1RA; being common to all 4 isoforms of IL1RA (US2007027082 [0115]).
The present invention discloses further peptide fragments of IL-1RA; in one embodiment being as short as 10 amino acids and in one embodiment comprising or consisting of SGRKSSKMQA (SEQ ID NO:1). Such fragments are shown herein to directly bind to IL-1RI and interfere with the binding of IL-1R to IL-1 beta; which is in contrast to the 35-amino acid long peptide fragment (“SEQ ID NO:13”) of US2007027082 that does not bind IL1R1.
Short peptides according to the present invention may comprise or consist of SGRKSSKMQA (SEQ ID NO:1) or variants, fragments, or variants of fragments thereof. They have the advantage over both full-length IL1RA (anakinra) and the 35 and 42 amino-acid long peptides of US2007027082 (both comprising the subsequence SGRKSSKMQA) that they are very stable, has a high solubility and also have a low cost of synthesis. These effects occur with a retained ability of the peptides to bind IL1R1 and antagonise the effect of IL-1.
Further short peptides of the present invention derived from IL1RA invention comprise or consist of RIWDVNQKT (SEQ ID NO:29), TAMEADQPVS (SEQ ID NO:35) or GPNAKLEEKA (SEQ ID NO:36) or variants, fragments, or variants of fragments thereof; having the same advantages as outlined for SEQ ID NO:1 herein.
Furthermore, peptides of a certain short length; such as the 10-amino acid peptide of SEQ ID NO:1, have an increased capability of passing the blood-brain-barrier (BBB) to elicit an effect on cells of the central nervous system (CNS); thus enabling use of said short peptides on neuroinflammatory disorders associated with IL-1. An effect on neurons of IL1RA or peptide fragments thereof has not been addressed in the art previously, nor has passing the BBB. A positive effect on neurite outgrowth and neuronal cell survival is shown herein.